The advent of genetically modified crops holds the promise of improving crop yield and productivity as well as reducing the number of pesticides and other toxic compounds needed for crop success. These benefits are conferred via the transformation of crop plants with new genes beneficial to growth or conveying disease or pathogen resistance. However, transformation events often result in the presence of ancillary genetic material that is not useful for the expression of the desired trait. These ancillary sequences are necessary for the transformation processes, but they do not positively contribute to the final cultivar and in fact lessen its desirability to the consumer. The presence of these undesirable sequences may also complicate the regulatory procedures necessary to bring the cultivar to the market place. A reliable method for eliminating the unwanted ancillary sequences would thus improve commercial viability by increasing public acceptance and simplify the regulatory process. Additionally, it will be useful to have certain traits only present during intermediate generations and then removed in harvested generations. The prior art has not recognized the importance of these problems, nor has it worked to provide a solution.
Plants are increasingly being looked to as platforms for the production of materials, foreign to plant systems. As the art of genetic engineering advances it will be possible to engineer plants for the production of a multiplicity of monomers and polymers, currently only available by chemical synthetic means. The accumulation of these materials in various plant tissues will be toxic at some level and it will be useful to tightly regulate the relevant genes to prevent expression in inappropriate plant tissues.
Currently few methods exist that provide for tightly regulated transgene expression. Non-specific expression of transgenes in non-target cells, tissues, or generation hinders plant transgenic work. This is important where the goal is to produce such high levels of materials in transgenic plants that may be phytotoxic or adversely affects normal plant development. Conditional transgene expression would enable economic production of desired chemicals, monomers, and polymers at levels likely to be phytotoxic to growing plants by restricting their production to production tissue of transgenic plants either just prior to or after harvest of the crop biomass used for extracting the desired product. Therefore, lack of a commercially usable conditional expression system and the difficulty in attaining a reliable, high-level expression both limit development of transgene expression in plants.
Conditional or regulated expression has been reported in plants (see De Veylder, L. et al., Plant Cell Physiol. 38:568–577 (1997); Gatz, C., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89–108 (1997); Hansen, G. et al., Mol. Gen. Genet. 254:337–343 (1997); Jepson, I., PCT Int. Appl. WO 9706269 A1 (1997), Jepson, I, et al. PCT Int. Appl. WO 9711189 A2 (1997), and other references within this application). However, when tested stringently for basal non-specific expression, very few have been strictly specific (Odell J. T. et al., Plant Physiol. 106:447–458 (1994); van der Geest et al., Plant Physiol., 109(4), 1151–58 (1995); Ma et al., Aust. J. Plant Physiol., 25(1), 53–59 (1998); Czako et al., Mol. Gen. Genet., 235(1), 33–40 (1992)). Such promoters are not suitable for some applications, such as the use of transgenes for expressing novel phytotoxic proteins, enzymes that lead to the biosynthesis of phytotoxic products, and/or gene silencing. Site-specific recombination in plants (Odell et al., Plant Physiol. 106:447–458 (1994); Odell et al., PCT Int. Appl. WO 9109957 (1991); Surin et al., PCT Int. Appl WO 9737012 (1997)) and the reduction in the proficiency of Cre-mediated recombination by mutant lox P sites and their use in increasing the frequency of Cre-lox based integration have been reported (Albert et al., Plant J. 7:649–59 (1995); Araki et al., Nucleic Acids Res. 25:868–872 (1997′)). However, the use of the mutant sites to enhance the specificity Cre-mediated recombination in conjunction with chimeric Cre genes under the control of available regulated promoters has not been demonstrated. Thus, there is a need for an appropriately stringent, site-specific recombination system for a commercially-attractive, conditional site-specific recombination.
Directed excision of a transgene from the plant genome has been reported using recombinase specific-sites and a recombinase. In Russel et al. (Mol. Gen. Genet. 234:49–59 (1992)), utility of these techniques is evaluated to remove a selectable marker for transformation from the plant, while leaving a preferred non-selectable trait. Variation in efficiency of excision of transgenes in different plants was also examined and comparison was made between introduction of the cre gene by re-transformation or by cross pollination. However, incorporation of promoters for conditional or regulated expression was not attempted.
Ow et al. (PCT Int. Appl. WO 9301283 A1 (1992)) also examine directed excision of selectable markers for transformation from plants, while leaving a preferred non-selectable trait that could be operably linked to control sequences capable of effecting the timing of said expression in higher plants. Their disclosed technique is limited by the requirement that the recombinase for excision must be introduced via a second round of transformation either directly to the initially transformed plants or by cross pollination of the independently regenerated plants from first and second round transformants.
In one disclosure, Oliver et al. (U.S. Pat. No. 5,977,441) demonstrate limited expression of a desired introduced gene in a transgenic plant, according to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location. This is achieved via: 1) the insertion of a transiently-active promoter in functional relation with a structural gene whose expression results in an altered plant phenotype, but separated by a blocking sequence that is flanked by specific excision sequences; and 2) a second DNA sequence that comprises an inducible promoter operably linked to a gene encoding a site-specific recombinase. These methods ultimately permit the expression of certain plant traits to be under external control by application of an external stimulus, through hybridization, or by direct introduction of a recombinase into a plant. However, Oliver et al. did not consider the need for eliminating unwanted ancillary sequences, such as selection markers.
Surin et al. (PCT Int. Appl. WO 9737012 A1 (1997)) disclose a method whereby single-step excision of transgenes is achieved, such as selectable marker genes or reporter genes, from genetically-transformed organisms. This is possible by the incorporation of both a recombinase genetic unit (comprising a promoter, recombinase gene, and terminator) and a transgene unit (comprising a promoter, transgene, and terminator) within a single expression cassette, all flanked by recombinase sites. This genetic unit facilitates multiple sequential genetic transformation events using the same selectable marker gene and provides means for tightly regulating transgene expression in genetically-manipulated plants. Additionally, the need for multiple transformation events or sexual crossing to produce a single cell comprising both the loci for DNA recombination and the site-specific recombinase is alleviated, since their method facilitates precise excision of genetic material in a single generation using promoters that are differentially activated. Although Surin et al. allow for the incorporation of a second separate expression cassette, their techniques are limited in that a maximum of two transgenic units may be expressed within the plant at different time periods, the first of which must be removed prior to the expression of the second. At no point may the second transgene be removed from the genome if desired.
Hodges et al. (U.S. Pat. No. 6,110,736) describe a method based on homologous recombination which enables the targeting of a length of DNA to a specific non-lethal site in the host's genome, and provides for the removal of any randomly inserted DNA sequences, using site-specific recombinase sites and the corresponding recombinase protein. The need met through the application of the site-specific recombinase system for gene excision is specifically to maintain control over the copy number and the location of the inserted DNA. A further embodiment of the invention combines use of cre/lox and FLP/FRT to ultimately leave only the desired DNA sequences integrated into the chromosome while the selectable marker is removed from the chromosome. However, this work relies is confined to instances where precise homologous recombination can be executed, which necessarily requires precise engineering of the vector sequences to match sequences within the chromosome.
Finally, Perez and Flament (PCT Int. Appl. WO 9838323 (1998)) discuss the insertion of a of a male sterility gene (AMS) to create a cytoplasmic or nuclear male sterile plant. This plant avoids the dissemination of pollen, and therefore necessarily prevents dissemination of any transgenes that are linked to the male sterility genes. In further embodiments of their invention, the AMS gene can serve as a positive marker for the screening of plants having integrated a transgene of interest; or, the gene, when genetically linked to a transgene, can be excised using a system of transposition (e.g. systems of recombination such as Cre/lox or FLP/FRT). Limitations of these techniques focus on the requirement that a transgene must be directly linked to a transgene, thereby requiring linked expression. No allowance is made for conditional expression, whereby expression of one gene may activate, or excise, another later in the life cycle.
The methods described above are useful for removal of transgenes and ancillary genetic material from plants but are limited in their ability to regulate transgene expression at various times in a plant life cycle or in hybrid progeny. The problem to be solved therefore is to develop a method for the tightly regulated conditional expression and removal of genetic traits in plants, both in initial transformants and in hybrid.
Applicant has solved the stated problem by providing developmentally regulated germ-like promoters that allow using at least two different recombinases for the selective expression and/or removal of genetic traits.